HIF-1α Regulates TGF-β1in Mediating Porcine Pulmonary Arterial Endothelial Cells Cultured At Hypoxia To Mesenchymal Transition

【Objective】To observe the changes of porcine pulmonary arteries endothelial cellsin morphology and physiological characeristics,and the effect of the endothelial cells’migration ability in hypoxia circumstance;To investigate the relationship betweenHIF-1α and TGF-β1,and the role of them in mediation of endothelial-to-mesenchymaltransition.【Methods】The study was composed of two parts：(1)The part of cell withouttransfected plasmid：The generation of pig’s pulmonary arteries endothelial cellsused the enzyme digested method，the scratch wound assay was used to detect themigration ability of endothelial cells in hypoxia environment,And set the normoxiagroup as a control.Using Immunofluorescence to double labeling of endothelialcell-specific antigen VE-cadherin and mesenchymal cell-specific antigen α-SMAobservation,and observe that the expression levels of VE-cadherin and α-SMA innormoxia and hypoxia group, calculate the conversion rate for statistical analysis;(2)The part of cell transfected plasmid：To transfect HIF-1α plasmid into cells in order tooverexpressing it or silencing it.when the HIF-1α gene was overexpressed orsilenced，western blotting and RT-PCR techniques were used for detecting protein andits mRNA expression of VE-cadherin、TGF-β1and α-SMA in porcine pulmonaryarteries endothelial cells respectively．【Results】1. The part of cell without transfected plasmid：(1)Compared with normoxia group, the endothelial cells under hypoxia circumstancehas a alteration in cell morphology which from cobblestone-like structure tospindle-like structure; At the same time, primary porcine pulmonary arteries endothelial cells were shown to reduce intercellular adhesion and increase motilityunder hypoxia circumstance(P<0.01).(2) Immunofluorescence results showed that there have been changes in themorphological and biological characteristics when endothelial cells were in a hypoxicenvironment(P<0.01);(3) Western blotting results showed that protein levels of HIF-1α、TGF-β1andα-SMA were higher in the hypoxia group than that were in the nomorxia group（P<0.05）;However, protein levels of VE-cadherin was gradually reduced（P<0.05）.(4) RT-PCR results were the same as Western blotting results（P<0.05）.2. The part of cell transfected plasmid：(1) HIF-1α overexpression plasmids were transfected primary porcine pulmonaryartery endothelial cells(PAECs) successfully;Compared with other three groups’,theprotein and mRNA expression level of α-SMA in hypoxia4days+HIF-1αoverexpression plasmid group was increased(P＜0.05);while VE-cadherin protein andmRNA expression level was lower than other three groups’(P＜0.05).(2) HIF-1α interference plasmids were transfected primary porcine pulmonary arteryendothelial cells(PAECs) successfully;Compared with hypoxia4days+blank plasmidgroup’s,the protein and mRNA expression level of α-SMA in hypoxia4days+HIF-1αinterference plasmid group was induced(P＜0.05)，but it was still higher thannormoxia groups’;while VE-cadherin protein and mRNA expression level was higherthan hypoxia4days+blank plasmid group’s(P＜0.05)，but it was still lower thannormoxia groups’.(3) After HIF-1α overexpression plasmids were transfected primary porcinepulmonary artery endothelial cells(PAECs) successfully,the protein and mRNA levelsof TGF-β1was observably increased compared with other three groups’ in endothelialcells(P＜0.05);while HIF-1α interference plasmids were transfected primary porcinepulmonary artery endothelial cells(PAECs) successfully,the protein and mRNA levelsof TGF-β1was observably lower than hypoxia4days+blank plasmid group’s inendothelial cells(P＜0.05),and there was no difference than normoxia groups’ （P>0.05）.【Conclusions】(1) Primary cultured porcine pulmonary arteries endothelial cells had a morphologicalchange under hypoxia;(2) Hypoxia reduce primary porcine pulmonary artery endothelial cells’ intercellularadhesion and increase their migration ability, may be one of the mechanisms of theendothelial-to-mesenchymal transition;(3) Hypoxia could stabilize HIF-1α protein in the nuclear,then HIF-1α up-regulationthe level of expression of TGF-β1in endothelial cells,and TGF-β1promote theprocess of endothelial-to-mesenchymal transtion.